In Vitro Analysis of Hedgehog Acyltransferase and Porcupine Fatty Acyltransferase Activities.

Hedgehog and Wnt proteins are modified by covalent attachment of the fatty acids palmitate and palmitoleate, respectively. These lipid modifications are essential for Hedgehog and Wnt protein signaling activities and are catalyzed by related, but distinct fatty acyltransferases: Hedgehog acyltransferase (Hedgehog) and Porcupine (Wnt). In this chapter, we provide detailed methods to directly monitor Hedgehog and Wnt protein fatty acylation in vitro. Palmitoylation of Sonic hedgehog (Shh), a representative Hedgehog family member, is assayed using purified Hedgehog acyltransferase (Hhat) or Hhat-enriched membranes, a recombinant 19 kDa Shh protein or C-terminally biotinylated Shh 10-mer peptide, and I-iodopalmitoyl CoA as the donor fatty acyl CoA substrate. The radiolabeled reaction products are quantified by SDS-PAGE and phosphorimaging or by γ-counting. To assay Wnt acylation, the reaction consists of a biotinylated, double disulfide-bonded Wnt peptide containing the sequence surrounding the Wnt3a acylation site, [I] iodo-cis-9-pentadecenoyl CoA, and Porcupine-enriched membranes. Radiolabeled, biotinylated Wnt3a peptide is captured on streptavidin coated beads and the reaction product is quantified by γ-counting.